Detection reagent or kit for detecting RS virus and method for detecting RS virus

Abstract

To provide an inspection reagent, a kit, and a method capable of detecting RS viruses with high sensitivity.SOLUTION: An RS virus detection reagent or a kit includes an anti-RS virus N protein monoclonal antibody containing a heavy chain VDJ region containing an amino acid sequence represented by sequence number 1, a light chain VJ region containing an amino acid sequence represented by sequence number 2, and / or a light chain VJ region containing an amino acid sequence represented by sequence number 3, or an antigen binding fragment thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a detection reagent or kit for detecting RS virus and a method for detecting RS virus. [Background technology]

[0002] Monoclonal antibodies recognize only specific antigens, and are therefore widely used to detect those specific antigens. Respiratory syncytial virus (RSV) envelopes contain G protein, a glycoprotein, and F protein, which is involved in cell fusion (Non-Patent Document 1). G protein is known to have a large difference in amino acid sequence between RSV subtypes (types A and B) (Non-Patent Document 1). On the other hand, F protein is known to have a small difference in amino acid sequence between RSV subtypes (types A and B), and many RSV test kits detect F protein. However, it has been very difficult to develop an RSV test kit that shows a practical positive detection rate that can detect all clinical isolates simply by increasing the affinity of antibodies to F protein (Patent Document 1).

[0003] The N protein of RS virus is also called nucleoprotein and consists of 391 amino acid residues. The N protein is a component of a ribonucleoprotein complex called nucleocapsid, and surrounds the RS virus genomic RNA to form a helical structure. The N protein is known to have the smallest difference in amino acid sequence between RS virus subtypes (types A and B). However, it is very difficult to detect all clinical isolates using only antibodies against the N protein, and in order to develop an RS virus test kit that can detect all clinical isolates, it was necessary to mix antibodies against the F protein and the N protein (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6454274 [Non-patent literature]

[0005] [Non-Patent Document 1] Collins PL and Karron RA. Respiratory syncytial virus. In: Knipe DM et al., eds. Fields Virology, 6th ed. Lippincott Williams & Wilkins, Philadelphia: pp 1086-1123 (2013). Summary of the Invention [Problem to be solved by the invention]

[0006] Currently, various types of test reagents using anti-RS virus antibodies are commercially available. However, when RS virus is detected using these conventional test reagents, the sensitivity is insufficient.

[0007] In view of the above-mentioned current situation, an object of the present invention is to provide a test reagent capable of detecting RS virus with high sensitivity. [Means for solving the problem]

[0008] The inventors discovered that the detection sensitivity of RS virus can be improved by using an antibody against the N protein (nucleoprotein) of RS virus in order to efficiently capture RS virus contained in a sample at the determination site and increase the sensitivity of the immunochromatography device, and thus completed the present invention.

[0009] That is, the present invention is as follows. [1] A heavy chain VDJ region comprising the amino acid sequence represented by SEQ ID NO:1, or a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence represented by SEQ ID NO:6, an HCDR2 comprising the amino acid sequence represented by SEQ ID NO:7, and an HCDR3 comprising the amino acid sequence represented by SEQ ID NO:8, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:1, and having binding activity to RS virus N protein; and a light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:2, or a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence represented by SEQ ID NO:9, an LCDR2 comprising the amino acid sequence represented by SEQ ID NO:10, and an LCDR3 comprising the amino acid sequence represented by SEQ ID NO:11, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:2, and having binding activity to RS virus N protein; and / or a light chain VJ region comprising the amino acid sequence represented by SEQ ID NO: 3, or an LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 13, and an LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 14, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, and having binding activity to RS virus N protein; A reagent or kit for detecting RS virus, comprising a first anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof. [2] The RS virus detection reagent or kit described in [1], which is a sandwich-type immunoassay reagent comprising a capture molecule and a detection molecule, wherein the capture molecule is the first anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof. [3] The reagent or kit for detecting RS virus described in [2], wherein the detection molecule is the first anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof. [4] The detection molecule comprises a heavy chain VDJ region comprising the amino acid sequence represented by SEQ ID NO: 4, or an HCDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, an HCDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and an HCDR3 comprising the amino acid sequence represented by SEQ ID NO: 15, and an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4, and having binding activity to RS virus N protein; and A light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:2, or a light chain VJ region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO:10, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO:11, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:2, and having binding activity to RS virus N protein; and / or a light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:5, or an LCDR1 comprising the amino acid sequence represented by SEQ ID NO:12, an LCDR2 comprising the amino acid sequence represented by SEQ ID NO:13, and an LCDR3 comprising the amino acid sequence represented by SEQ ID NO:16, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:5, and having binding activity to RS virus N protein; The reagent or kit for detecting RS virus according to [2], wherein the second anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof comprises: [5] The reagent or kit for detecting RS virus according to any one of [1] to [4], wherein the antigen-binding fragment is a peptide fragment selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dsFv, diabody and minibody. [6] The reagent or kit for detecting RS virus according to any one of [1] to [5], which is a reagent or kit for immunochromatography. [7] A heavy chain VDJ region comprising the amino acid sequence represented by SEQ ID NO:1, or a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence represented by SEQ ID NO:6, an HCDR2 comprising the amino acid sequence represented by SEQ ID NO:7, and an HCDR3 comprising the amino acid sequence represented by SEQ ID NO:8, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:1, and having binding activity to RS virus N protein; and a light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:2, or a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence represented by SEQ ID NO:9, an LCDR2 comprising the amino acid sequence represented by SEQ ID NO:10, and an LCDR3 comprising the amino acid sequence represented by SEQ ID NO:11, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:2, and having binding activity to RS virus N protein; and / or a light chain VJ region comprising the amino acid sequence represented by SEQ ID NO: 3, or an LCDR1 comprising the amino acid sequence represented by SEQ ID NO: 12, an LCDR2 comprising the amino acid sequence represented by SEQ ID NO: 13, and an LCDR3 comprising the amino acid sequence represented by SEQ ID NO: 14, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, and having binding activity to RS virus N protein; A method for detecting RS virus using a first anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof, comprising: [8] The method according to [7], which is an immunochromatographic method. Effect of the Invention

[0010] According to the RS virus detection reagent, RS virus detection kit, or RS virus detection method of the present invention, RS virus can be detected with high sensitivity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 1. How to detect RSV One embodiment of the present invention is a method for detecting RS virus. The method of this embodiment comprises detecting a heavy chain VDJ region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:1, or a heavy chain complementarity determining region (HCDR) 1 consisting of the amino acid sequence of SEQ ID NO:6, an HCDR2 consisting of the amino acid sequence of SEQ ID NO:7, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:8, and having an activity of binding to RS virus N protein; and a light chain VJ region comprising an amino acid sequence of SEQ ID NO:2, or a light chain complementarity determining region (LCDR) 1 consisting of the amino acid sequence of SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence of SEQ ID NO:10, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:11. and / or a light chain VJ region comprising an amino acid sequence represented by SEQ ID NO: 3, or a light chain VJ region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 12, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 13, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 14, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, and having binding activity to RS virus N protein. That is, the method of this embodiment is a method in which RS virus is used as a test subject, and a monoclonal antibody or an antigen-binding fragment thereof that recognizes a specific amino acid sequence of N protein as an epitope is used for detection of RS virus.

[0012] In the method of the present invention, an immunoassay is performed using a monoclonal antibody or an antigen-binding fragment thereof that recognizes N protein as an antigen. Here, "recognize" means to react specifically, i.e., to undergo an antigen-antibody reaction. "Specific" means that in a liquid system in which a protein and the antibody are mixed, the antibody does not undergo a detectable antigen-antibody reaction with the protein component of the antigen, or, even if it does undergo some kind of binding reaction or association reaction, it undergoes a reaction that is clearly weaker than the antigen-antibody reaction of the antibody with the antigen.

[0013] The method of the present embodiment may also use an antigen-binding fragment obtained by isolating only the antigen-binding site based on a monoclonal antibody. That is, the scope of the present embodiment also includes an embodiment in which a fragment having specific antigen-binding ability (antigen-binding fragment), such as Fab, Fab', F(ab')2, single-chain antibody (scFv), dsFv, diabody, or minibody, which binds to the N protein of RS virus and is produced by a known method, is used together with or instead of the monoclonal antibody. The class of the monoclonal antibody referred to here is not limited to IgG, and may be IgM or IgY.

[0014] The monoclonal antibody used in the method of the present embodiment can be obtained by immunizing an animal to be immunized with a complex or extract containing the antigen of interest, or the antigen or a partial peptide thereof, using a known immunological technique, and preparing a hybridoma using the cells of the animal to be immunized. The length of the peptide used for immunization is not particularly limited, but a peptide of preferably 5 amino acids or more, more preferably 10 amino acids or more can be used as an immunogen. The immunogen can be obtained from a culture medium, or can be obtained by incorporating DNA encoding an arbitrary antigen into a plasmid vector, introducing this into a host cell, and expressing it. An arbitrary antigen or a partial peptide thereof to be used as an immunogen can be expressed as a fusion protein with a protein such as those exemplified below, and can be used as an immunogen after purification or as it is. To prepare a fusion protein, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (TRX), Nus tag, S tag, HSV tag, FRAG tag, polyhistidine tag, etc., which are commonly used by those skilled in the art as "protein expression / purification tags", can be used. The fusion protein with these is preferably used as an immunogen after cleaving the antigen or its partial peptide portion and the remaining tag portion with a digestive enzyme, and separating and purifying it.

[0015] Preparation of monoclonal antibodies from immunized animals can be easily performed by the well-known method of Kohler et al. (Kohler et al., Nature, vol. 256, p. 495-497 (1975)). That is, antibody-producing cells such as spleen cells and lymphocytes are collected from the immunized animal, and these are fused with mouse myeloma cells by a conventional method to prepare hybridomas. The obtained hybridomas are cloned by limiting dilution or the like, and from the monoclonal antibodies produced by each cloned hybridoma, a monoclonal antibody that undergoes an antigen-antibody reaction with the antigen used to immunize the animal is selected.

[0016] Monoclonal antibodies can be purified from ascites or culture supernatant by known immunoglobulin purification methods, such as fractionation by salting out using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, and gel filtration. Purification can also be performed by affinity chromatography using a carrier bound to Protein A, Protein G, or Protein L, depending on the immunized animal species and the class of the monoclonal antibody.

[0017] The method of this embodiment is a method of measuring by immunoassay utilizing an antigen-antibody reaction between the monoclonal antibody or antigen-binding fragment thereof prepared as described above (hereinafter, in the description before the Examples, unless otherwise clear from the context, "antibody" means "monoclonal antibody or antigen-binding fragment thereof") and an antigen in a sample. As the immunoassay method for this purpose, any method well known to those skilled in the art, such as a competitive method, an agglutination method, a Western blot method, an immunostaining method, or a sandwich method, can be used. In this specification, "measurement" includes any of quantitative, semi-quantitative, and detection.

[0018] The antibody that recognizes at least one RS virus N protein used in the method of this embodiment (the first anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof, hereinafter also referred to as "Antibody A") has the following characteristics. A heavy chain VDJ region comprising the amino acid sequence represented by SEQ ID NO:1; and A light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:2, and / or A light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:3. Contains, or a heavy chain VDJ region which comprises, instead of a heavy chain VDJ region containing the amino acid sequence represented by SEQ ID NO:1, an HCDR1 consisting of the amino acid sequence represented by SEQ ID NO:6, an HCDR2 consisting of the amino acid sequence represented by SEQ ID NO:7, and an HCDR3 consisting of the amino acid sequence represented by SEQ ID NO:8, and which comprises an amino acid sequence having 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO:1, and which has binding activity to RS virus N protein; a light chain VJ region which comprises an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO:10, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO:11, instead of the light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:2, and which comprises an amino acid sequence having 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO:2, and has binding activity to RS virus N protein; and / or The light chain VJ region includes an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 12, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 13, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 14, instead of a light chain VJ region containing the amino acid sequence represented by SEQ ID NO: 3, and has an amino acid sequence that has 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 3, and has binding activity to RS virus N protein. That is, provided that the CDRs of each heavy and light chain are conserved and the binding activity to RS virus N protein is maintained, 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid may be deleted, substituted or added to the amino acid sequence of other regions. Such a protein having an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted or added to the amino acid sequence of SEQ ID NO: 1, 2 or 3 is substantially identical to the protein having the amino acid sequence of SEQ ID NO: 1, 2 or 3.

[0019] In this disclosure, "sequence identity" refers to a value calculated by comparing two sequences using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., default or initial setting parameters).

[0020] Antibody A used in the method of this embodiment may have two light chains with homologous sequences, or may have two light chains with different amino acid sequences.

[0021] The antibody A used in the method of this embodiment is composed of the above-mentioned heavy chain VDJ region and heavy chain constant region, and the above-mentioned light chain VJ region and light chain constant region. The heavy chain constant region is composed of three domains C H 1. C H 2 and C H The heavy chain constant region is an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. The light chain constant region is composed of one domain, C L The light chain constant region is a kappa or lambda constant region.

[0022] Antibody A used in the method of this embodiment can be produced, for example, by inserting the above-mentioned DNA encoding the heavy chain and DNA encoding the light chain into an expression vector, transforming a host cell with the vector, and culturing the host cell.

[0023] As the immunoassay, a sandwich-type immunoassay is preferred. In a sandwich-type immunoassay, a capture antibody (capture molecule) immobilized on a solid phase or the like and a labeled detection antibody (detection molecule) are usually used, and a complex is formed by sandwiching an antigen between these antibodies, and the complex is detected by the label. Sandwich-type immunoassays are well known in the field of immunoassays, and can be performed, for example, by immunochromatography or ELISA. The method of this embodiment may utilize any of the well-known sandwich-type immunoassays, except for using a monoclonal antibody that recognizes the above-mentioned N protein as an antigen.

[0024] In the sandwich immunoassay, as described above, one or more kinds of antibodies that recognize an antigen are used as the capture antibody and the detection antibody. The capture antibody and the detection antibody may be the same kind of antibody. In this case, for example, both the capture antibody and the detection antibody can be the above-mentioned antibody A.

[0025] Furthermore, the capture antibody and the detection antibody may be different antibodies. When two or more types of antibodies are used, at least one of these two types of antibodies must be antibody A. Preferably, the capture antibody is antibody A. Preferably, the capture antibody is antibody A, and the detection antibody is an antibody having the following characteristics (a second anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof, hereinafter also referred to as "antibody B"). A heavy chain VDJ region comprising the amino acid sequence represented by SEQ ID NO:4; and A light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:2, and / or A light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:5. Contains, or a heavy chain VDJ region which comprises an HCDR1 consisting of the amino acid sequence represented by SEQ ID NO:6, an HCDR2 consisting of the amino acid sequence represented by SEQ ID NO:7, and an HCDR3 consisting of the amino acid sequence represented by SEQ ID NO:15, instead of a heavy chain VDJ region containing the amino acid sequence represented by SEQ ID NO:4, and which comprises an amino acid sequence having 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO:1, and which has binding activity to RS virus N protein; a light chain VJ region which comprises an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO:10, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO:11, instead of the light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:2, and which comprises an amino acid sequence having 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO:2, and has binding activity to RS virus N protein; and / or The light chain VJ region includes an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 12, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 13, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 16, instead of a light chain VJ region including the amino acid sequence represented by SEQ ID NO: 5, and includes an amino acid sequence having 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 3, and has binding activity to RS virus N protein. That is, provided that the CDRs of each heavy and light chain are conserved and the binding activity to RS virus N protein is maintained, 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid may be deleted, substituted or added to the amino acid sequence of other regions. Such a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added to the amino acid sequence of SEQ ID NO: 1, 2 or 3 is substantially identical to a protein having the amino acid sequence of SEQ ID NO: 4, 2 or 5.

[0026] When used in the method of this embodiment, antibody B may have two light chains having homologous sequences, or may have light chains having two different amino acid sequences.

[0027] When used in the method of this embodiment, antibody B is composed of the above-mentioned heavy chain VDJ region and heavy chain constant region, and the above-mentioned light chain VJ region and light chain constant region. The heavy chain constant region is composed of three domains C H 1. C H 2 and C H The heavy chain constant region is an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. The light chain constant region is composed of one domain, C L The light chain constant region is a kappa or lambda constant region.

[0028] When used in the method of this embodiment, antibody B can be produced, for example, by inserting the above-mentioned DNA encoding the heavy chain and DNA encoding the light chain into an expression vector, transforming a host cell with the vector, and culturing the host cell.

[0029] In sandwich-type immunoassays, the solid phase on which the antibody is immobilized can be anything that can immobilize the antibody by known techniques, and can be selected from known materials such as a porous thin film (membrane) with capillary action, particulate material, test tube, and resin plate. In addition, the material that labels the antibody can be an enzyme, a radioisotope, a fluorescent material, a luminescent material, a colored particle, a colloid particle, and the like. Among the immunoassays using the various materials mentioned above, the immunochromatography method, which is a lateral flow immunoassay using a membrane, is preferred, particularly from the viewpoint of the simplicity and speed of clinical testing.

[0030] The method of this embodiment makes it possible to detect whether or not a subject is infected with RS virus, and when N protein is detected in a test sample, it can be determined that the subject is infected with RS virus.

[0031] When the method of this embodiment is used, RS virus can be specifically recognized. In particular, the antibody that recognizes the N protein of RS virus used in the method of this embodiment does not recognize other viruses, such as Adenovirus, Coxsackievirus, Echovirus, Herpes simplex virus, Human Metapneumovirus, Influenza virus, Measles virus, Mumps virus, and Parainfluenza virus, and does not erroneously detect these viruses.

[0032] Furthermore, according to the method of this embodiment, it is possible to detect clinical isolates that cannot be detected when an antibody that recognizes the F protein of RS virus is used.

[0033] The specimen sample in the method of this embodiment may be a pharyngeal or nasal swab, pharyngeal or nasal wash, nasal aspirate, saliva, serum, rectal swab, stool, stool suspension, urine, corneal swab, or the like.

[0034] 2. RS virus measurement reagents and kits Another embodiment of the present invention is a reagent and kit for measuring RS virus. The reagent and kit of this embodiment include a heavy chain VDJ region comprising an amino acid sequence represented by SEQ ID NO:1, or a heavy chain complementarity determining region (HCDR) 1 consisting of the amino acid sequence represented by SEQ ID NO:6, an HCDR2 consisting of the amino acid sequence represented by SEQ ID NO:7, and an HCDR3 consisting of the amino acid sequence represented by SEQ ID NO:8, and having an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:1 and having binding activity to RS virus N protein; and a light chain VJ region comprising an amino acid sequence represented by SEQ ID NO:2, or a light chain complementarity determining region (LCDR) 1 consisting of the amino acid sequence represented by SEQ ID NO:9, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO:10, and an HCDR3 consisting of the amino acid sequence represented by SEQ ID NO:11. and / or a light chain VJ region comprising an amino acid sequence represented by SEQ ID NO: 3, or a light chain VJ region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 12, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 13, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 14, an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, and a light chain VJ region having binding activity to RS virus N protein; and / or a light chain VJ region comprising an amino acid sequence represented by SEQ ID NO: 3, or a light chain VJ region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO: 12, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO: 13, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO: 14, an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3, and a light chain VJ region having binding activity to RS virus N protein. The reagent and kit of this embodiment is, unless otherwise specified and unless otherwise contradicted, a reagent and kit for use in the method described in Section "1. Method for detecting RS virus". ... In this disclosure, a "reagent" and a "kit" are referred to as a "reagent" when it contains only one component for carrying out the method of the present invention, and a "kit" when it contains multiple components, but no other distinction is made between them.

[0035] The reagent and kit of this embodiment may include an immunoassay device capable of performing a lateral flow immunoassay using a monoclonal antibody that recognizes the N protein as an antigen. The configuration of the immunoassay device will be described below as an example, but it is not intended that this embodiment be limited to the following.

[0036] The immunoassay device is composed of a support having a detection region where an antibody (capture antibody) that captures the measurement target (antigen) is immobilized, a label region having a mobile labeled antibody (detection antibody), a sample pad onto which a specimen is dropped, an absorption band that absorbs the developed specimen liquid, and a backing sheet for bonding these components together, and at least one of the capture antibody and detection antibody is the above-mentioned antibody A. This immunoassay device is also called an immunochromatographic test piece.

[0037] The support is a material capable of immobilizing an antibody (capture antibody) for capturing a substance to be detected (antigen), and also capable of not hindering the horizontal passage of liquid. It is preferably a porous thin film having capillary action, and is a material capable of transporting liquid and components dispersed therein by absorption. The material constituting the support is not particularly limited, and examples thereof include cellulose, nitrocellulose, cellulose acetate, polyvinylidene difluoride (PVDF), glass fiber, nylon, polyketone, and the like. Of these, a thin film made of nitrocellulose is more preferable. A membrane with an antibody immobilized thereon is called an antibody-immobilized membrane.

[0038] The label region is made of a porous substrate containing a labeled antibody (detection antibody), and the substrate may be made of commonly used materials such as glass fiber or nonwoven fabric. The substrate is preferably in the form of a pad having a thickness of about 0.3 mm to 0.6 mm so that a large amount of detection antibody can be impregnated therein. A porous substrate impregnated with detection antibody and dried is also called a dry pad.

[0039] Enzymes such as alkaline phosphatase and horseradish peroxidase, metal colloids such as gold colloids, silica particles, cellulose particles, colored polystyrene particles, and colored latex particles are often used to label the detection antibodies. When colored particles such as metal colloid particles, colored polystyrene particles, and colored latex particles are used, the labeling reagents aggregate to cause coloration, and this coloration is measured. Particles with antibodies immobilized thereon are called antibody-immobilized particles. The amount of antibody immobilized is not particularly limited, but it is sufficient that the antibody is present in the labeled area in an amount of several ng to several tens of μg.

[0040] The detection region refers to a part of a support where an antibody (capture antibody) that captures a substance to be detected (antigen) is immobilized. The detection region is provided with at least one region where an antibody (detection antibody) for capturing an antigen is immobilized. The detection region only needs to be included in the support, and the antibody can be immobilized on the support. The amount of antibody immobilized is not particularly limited, but it is sufficient to immobilize several ng to several tens of μg in the detection region.

[0041] The sample pad is a portion for adding a drop of a specimen, and is made of a porous material. The sample pad is the most upstream portion of the immunoassay device. Commonly used materials such as filter paper, glass fiber, and nonwoven fabric can be used for the sample pad. In order to use a large amount of specimen for the immunoassay, the pad is preferably about 0.3 mm to 1 mm thick. The specimen also includes a sample prepared using the specimen, such as a sample obtained by suspending the specimen in another solution.

[0042] The absorption band is a member for absorbing components that are supplied to the support and do not participate in the reaction in the detection area. The material may be a filter paper or sponge with high water retention made of a general natural or synthetic polymer compound, but a material with high water absorption is preferable in order to promote the development of the sample.

[0043] The backing sheet is a member for attaching and fixing all of the above-mentioned materials, i.e., the support, sample pad, label region, absorption band, etc., with partial overlap. The backing sheet is not necessarily required if these materials are arranged and fixed at optimal intervals, but it is generally preferable to use it from the viewpoint of convenience in manufacturing and use.

[0044] The immunoassay device may further include a control display area (component). The control display area is a portion that indicates that the test has been performed correctly. For example, the control display area is downstream of the detection area, and emits a signal by coloring or the like when a specimen sample passes through the detection area and reaches the control display area. The control display area may have immobilized thereon a substance that binds to the antibody bound to the labeled carrier, or may have immobilized thereon a reagent such as a pH indicator that changes color when the specimen reaches the control display area. When the antibody bound to the labeled carrier is a mouse monoclonal antibody, an anti-mouse IgG antibody may be used.

[0045] The size of the immunoassay instrument is not limited, but for example, the length is about several centimeters to a dozen centimeters, and the width is about several millimeters to several centimeters.

[0046] The immunoassay instrument may be housed in a storage container, which can prevent deterioration due to, for example, ultraviolet light or moisture in the air. Furthermore, when a contaminating or infectious specimen is used, the storage container can prevent the tester performing the assay from becoming contaminated or infected. For example, a resin case of an appropriate size can be used as the storage container, and the instrument can be stored in the case. The storage container and the immunoassay instrument housed therein may be collectively referred to as an immunoassay device.

[0047] In a method using an immunoassay device, a complex of a detection antibody capable of binding to a substance to be detected (labeled reagent) labeled with an appropriate labeling substance such as colored polystyrene particles or gold colloid is developed and moved on a solid support on which a capture antibody is immobilized, utilizing capillary action. As a result, a complex of capture antibody-substance to be detected-detection antibody is formed on the solid support, and the substance to be detected can be detected by detecting a signal derived from the label emitted from the complex (in the case of gold colloid, the part of the solid support on which the capture antibody capable of binding to the substance to be detected is immobilized turns red). The immunoassay method can be carried out at 5 to 35°C, preferably at room temperature.

[0048] The number of detection regions and the type of detection antibody contained in the label region are not limited to one, and by using antibodies corresponding to multiple objects to be measured, two or more antigens can be detected with the same immunoassay device.

[0049] The reagent or kit of this embodiment may include the immunoassay instrument. The kit may also include a blotter, a specimen collection instrument, and the like. EXAMPLES

[0050] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0051] Example 1: Preparation of anti-RS virus N protein monoclonal antibody 1. Preparation of Respiratory Syncytial Virus N Protein Antigen Respiratory syncytial virus (RSV) was infected into susceptible mammalian cells, which were then cultured for several days, after which the culture medium of the RSV-infected cells was inactivated by exposure to ultraviolet light.

[0052] 2. Preparation of anti-RS virus N protein monoclonal antibodies BALB / c mice were immunized with the inactivated RS virus antigen mentioned in 1., and the iliac lymph nodes were removed from the mice after they had been raised for a certain period of time. Using the "mouse iliac lymph node method" (Sado Y et al., Acta Histochem. Cytochem. 39: 89-94 (2006)), several hybridoma cell lines producing anti-RS virus N protein antibodies were obtained.

[0053] The obtained cell line was intraperitoneally administered to pristane-treated BALB / c mice, and antibody-containing ascites was collected about two weeks later. IgG was purified from the obtained ascites by affinity chromatography using a protein A column, and seven clones (No. 1 to 7) of purified anti-RS virus N protein monoclonal antibodies (hereinafter sometimes referred to as "anti-N protein antibodies") were obtained.

[0054] Example 2 Preparation of RS virus N antibody-immobilized membrane and RS virus N protein antibody-immobilized particles 1. Immobilization of anti-RS virus N protein antibody onto nitrocellulose membrane The anti-RS virus N protein antibodies Nos. 1 and 3 to 7 prepared in Example 1 were diluted with a buffer solution, and anti-mouse IgG antibody was prepared, and the anti-N protein antibody was applied in a line shape to the sample pad side of a nitrocellulose membrane backed with a PET film, and the anti-mouse IgG antibody was applied in a line shape to the absorber side. The nitrocellulose membrane was then thoroughly dried under hot air to obtain an anti-N protein antibody-immobilized membrane.

[0055] 2. Immobilization of anti-RS virus N protein antibody onto colored polystyrene particles The anti-N protein antibodies Nos. 2 to 7 prepared in Example 1 were each covalently bound to colored polystyrene particles, suspended in a suspension, and thoroughly dispersed by ultrasonic treatment to obtain anti-N protein antibody-bound colored polystyrene particles, which are referred to herein as anti-N protein antibody-immobilized particles.

[0056] Example 3 Comparative sensitivity test of sandwich immunoassay systems using RS virus antigens A specimen suspension containing antigen was prepared by mixing 30 μL of RS virus antigen solution with 400 μL of specimen suspension. The specimen suspension was then diluted 4-fold and 8-fold to prepare antigen-containing suspensions. Anti-N protein antibody immobilized particle suspensions were prepared by suspending anti-N protein antibody immobilized particles (No. 2 to 7) in the specimen suspension at a concentration of approximately 0.3%. No. 7 antibody immobilized particle was not used in subsequent tests due to its poor dispersibility. 4 μL of the anti-N protein antibody immobilized particle suspension was dropped into a 96-well plate per well.

[0057] 100μL of the antigen-containing suspension diluted 4-fold or 8-fold was dropped per well and pipetted. In addition, 100μL of the antigen-free sample suspension was dropped per well as a blank and pipetted. Each anti-N protein antibody immobilized membrane (No. 1, 3-9) was inserted so that one end of the membrane was immersed in each well, and after 8 minutes it was removed and the coloring state was evaluated.

[0058] The results were judged visually. If coloration from the anti-N protein antibody immobilized particles was visible on the nitrocellulose membrane where the anti-N protein antibody was applied in a line, the result was judged positive. If coloration was not visible, the result was judged negative. In addition, the positive result was judged on a five-point scale from + to +++++ based on the color intensity. The results are shown in Table 1.

[0059] [Table 1]

[0060] As shown in Table 1, strong coloration was observed in the system using No. 3 as the antibody-immobilized membrane and No. 3 or No. 4 as the antibody-immobilized particles. This demonstrated that in terms of detection sensitivity, using antibody No. 3 as the capture antibody and antibody No. 3 or No. 4 as the detection antibody is advantageous in detecting RS virus.

[0061] Example 4 Preparation of RS virus test device 1. Coating and drying of anti-RS virus N protein antibody-bound colored polystyrene particles A predetermined amount of the antibody-immobilized particles No. 4 prepared in Example 2-2 was applied to a glass fiber nonwoven fabric and thoroughly dried in hot air. In this specification, this is referred to as a label pad.

[0062] 2. Preparation of RS virus testing device The antibody-immobilized membrane No. 3 prepared in Example 2-1 and the labeled pad prepared in Example 4-1 were combined, attached to other components (backing sheet, absorption band, sample pad), and cut to a width of 5 mm to form an RS virus testing device.

[0063] 3. Confirmation of the specificity and accuracy of RS virus testing devices 50 μL of a sample suspension containing a virus that causes respiratory infection (10 mM Tris (pH 8.0), 1% (w / v) polyoxyethylene octylphenyl ether, 3% (w / v) arginine, 3% (w / v) BSA) was added dropwise to the RS virus testing device prepared in 2 and allowed to stand for 5 minutes.

[0064] If color development could be visually confirmed at both the anti-mouse IgG antibody and anti-N protein antibody application positions, it was judged as +. If color development could be visually confirmed only at the anti-mouse IgG antibody application position, but not at the anti-N protein antibody application position, it was judged as -. If color development could not be visually confirmed at the anti-mouse IgG antibody application position, it was judged as invalid. The results are shown in Table 2.

[0065] [Table 2]

[0066] As shown in Table 2, the immunoassay device using anti-RS virus N protein antibodies No. 3 and No. 4 reacted to RS virus but did not show cross-reactivity to other causative viruses of respiratory tract infections, confirming that it reacted specifically to RS virus.

[0067] Example 5: Analysis of the variable regions of anti-RS virus N protein monoclonal antibodies 1. Amino acid sequence analysis of the heavy chain VDJ region and light chain VJ region of the anti-N protein antibody The hybridoma cell lines producing the anti-N protein antibodies No. 3 and No. 4 prepared in Example 1 were sent to Takara Bio Inc., and a request was made to analyze the base sequences of the heavy chain VDJ region and the light chain VJ region of each antibody. Takara Bio Inc. extracted RNA from the hybridoma cell lines, and obtained base sequences from the amplification products of the anti-N protein antibody variable regions obtained by 5'-RACE PCR.

[0068] 2. Prediction of amino acid sequences of the variable regions of anti-N protein antibodies From the nucleotide sequence information obtained in 1, the amino acid sequences of the heavy chain VDJ region and the light chain VJ region, as well as the complementarity determining regions (CDRs) in each region, were predicted using IMGT / V-QUEST, a nucleotide sequence analysis tool of the IMGT (registered trademark) database. Table 3 shows the results for antibody No. 3, and Table 4 shows the results for antibody No. 4.

[0069] [Table 3]

[0070] [Table 4]

[0071] Example 6 Comparison of sensitivity of anti-RS virus N protein antibodies The detection sensitivity of an antigen detection reagent using the anti-N protein antibody prepared in Example 4 was compared with that of an antigen detection reagent using a commercially available anti-F protein antibody for three strains of RS virus isolated from clinical specimens and cultured.

[0072] Three strains of RS virus isolated from clinical specimens collected in 2016 were inoculated and grown in Vero cells. A two-fold dilution series of RS virus-infected cell culture supernatant was prepared using buffer, and a specified amount of each was added to the specimen suspension to prepare the sample. The results are shown in Table 5.

[0073] [Table 5]

[0074] A comparative sensitivity test was conducted using three virus strains isolated from clinical specimens. As a result, for two of the three strains, the antigen detection reagent using anti-N protein antibody showed sensitivity equal to or greater than that of the antigen detection reagent using anti-F protein antibody.

[0075] For one of the three strains, no reaction was observed at all with the antigen detection reagent using anti-F protein antibody, whereas high detection sensitivity was observed with the antigen detection reagent using anti-N protein antibody. [Industrial Applicability]

[0076] The reagent, kit and method of the present invention can be used in the medical field and in the field of manufacturing and selling in vitro diagnostic pharmaceuticals.

Claims

1. a heavy chain VDJ region comprising the amino acid sequence represented by SEQ ID NO:1, or a heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence represented by SEQ ID NO:6, an HCDR2 comprising the amino acid sequence represented by SEQ ID NO:7, and an HCDR3 comprising the amino acid sequence represented by SEQ ID NO:8, the heavy chain VDJ region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:1, and having binding activity to RS virus N protein; and a light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:3, or a light chain VJ region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO:12, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO:13, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO:14, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:3, and having binding activity to RS virus N protein; A reagent or kit for detecting RS virus, comprising a first anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof.

2. The reagent or kit for detecting RS virus according to claim 1, which is a sandwich-type immunoassay reagent comprising a capture molecule and a detection molecule, and the capture molecule is the first anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof.

3. The reagent or kit for detecting RS virus according to claim 2 , wherein the detection molecule is the first anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof.

4. the detection molecule comprises a heavy chain VDJ region comprising the amino acid sequence represented by SEQ ID NO:4, or an HCDR1 consisting of the amino acid sequence represented by SEQ ID NO:6, an HCDR2 consisting of the amino acid sequence represented by SEQ ID NO:7, and an HCDR3 consisting of the amino acid sequence represented by SEQ ID NO:15, and an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:4, and having binding activity to RS virus N protein; and a light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:5, or a light chain VJ region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO:12, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO:13, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO:16, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:5, and having binding activity to RS virus N protein; The reagent or kit for detecting RS virus according to claim 2, wherein the second anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof comprises:

5. The antigen-binding fragment is Fab, Fab', F(ab') ２ 2. The reagent or kit for detecting RS virus according to claim 1, wherein the antibody is a peptide fragment selected from the group consisting of a single chain antibody (scFv), a dsFv, a diabody and a minibody.

6. The RS virus detection reagent or kit according to claim 1, which is an immunochromatographic reagent or kit.

7. a heavy chain VDJ region comprising the amino acid sequence represented by SEQ ID NO:1, or a heavy chain VDJ region comprising an HCDR1 consisting of the amino acid sequence represented by SEQ ID NO:6, an HCDR2 consisting of the amino acid sequence represented by SEQ ID NO:7, and an HCDR3 consisting of the amino acid sequence represented by SEQ ID NO:8, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:1, and having binding activity to RS virus N protein; and a light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:3, or a light chain VJ region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO:12, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO:13, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO:14, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:3, and having binding activity to RS virus N protein; A method for detecting RS virus using a first anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof, comprising:

8. The method according to claim 7, which is an immunochromatographic method.

9. A heavy chain VDJ region comprising an amino acid sequence represented by SEQ ID NO:1, or a heavy chain complementarity determining region (HCDR) 1 consisting of the amino acid sequence represented by SEQ ID NO:6, an HCDR2 consisting of the amino acid sequence represented by SEQ ID NO:7, and an HCDR3 consisting of the amino acid sequence represented by SEQ ID NO:8, said heavy chain VDJ region comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:1, and having binding activity to respiratory syncytial virus N protein; and a light chain VJ region comprising the amino acid sequence represented by SEQ ID NO:3, or a light chain VJ region comprising an LCDR1 consisting of the amino acid sequence represented by SEQ ID NO:12, an LCDR2 consisting of the amino acid sequence represented by SEQ ID NO:13, and an LCDR3 consisting of the amino acid sequence represented by SEQ ID NO:14, and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:3, and having binding activity to RS virus N protein; An anti-RS virus N protein monoclonal antibody or an antigen-binding fragment thereof comprising:

Images